Estrogen-dependent activation of NCOA3 couples with p300 and NF-κB to mediate antiapoptotic genes in ER-positive breast cancer cells

Circumvention of apoptosis by the elevation of antiapoptotic proteins is an important cause of carcinogenesis. The induction of antiapoptotic genes, including B-cell CLL/lymphoma 2 (BCL2), BCL2 related protein A1 (BCL2A1), BCL2 like 1 (BCL2L1), BCL2L2, and myeloid cell leukemia 1 (MCL1), has been observed in multiple cancers, including breast cancer. However, the underlying mechanisms of their overexpression are still being investigated. Here, we revealed that BCL2, BCL2A1, BCL2L2, and MCL1 but not BCL2L1 were overexpressed in estrogen receptor (ER)-positive breast cancer cells and clinical biopsies. Stimulation with estrogen in ER-positive cell lines resulted in a dose-dependent increase in BCL2, BCL2A1, BCL2L2, and MCL1 mRNA levels. Molecular investigation revealed that nuclear factor kappa B (NF-κB) recruited histone acetyltransferase p300 and nuclear receptor coactivator 3 (NCOA3) to form a transcriptional complex. This complex docked the promoters of BCL2, BCL2A1, BCL2L2, and MCL1 and activated their expression. Interestingly, estrogen exposure dose-dependently activated NCOA3. Depletion of the NCOA3-p300-NF-κB components or blockage of NCOA3 function with inhibitors (gossypol and bufalin) in ER-positive cells suppressed BCL2, BCL2A1, BCL2L2, and MCL1 expression, while also decreasing cell viability, colony formation, cell invasion, and tumor growth. Collectively, our results demonstrate an upstream signaling that activates four antiapoptotic genes in ER-positive breast cancer cells. Importantly, our results also imply that targeting NCOA3 or blocking the assembly of the NCOA3-p300-NF-κB complex may be promising therapeutic strategies for treating ER-positive breast cancer. Supplementary Information The online version contains supplementary material available at 10.1007/s12672-023-00635-0.

were performed and results represented means of three replicates ± SD. Significant difference was determined by the One-way ANOVA, followed by Tukey's post-hoc test. ns: no significant difference; * P < 0.05 and ** P < 0.01. (C) The protein levels of ER and antiapoptotic proteins. Total protein extracts from cells in (A) were used for immunoblots to measure the protein levels of ERa,ERb,Bcl2A1,Bcl2,Mcl1,and GAPDH (loading control). Three independent replicates were performed. For each lane in a replicate, three independent protein samples were mixed with equal weights (20 µg for each). One group of immunoblot images were shown.
6 Figure S4. The quantified protein levels of ER and antiapoptotic proteins in HME1,

T47D and BT549 cells treated with or without E2
Protein signals in Figure S3C were quantified using Image J software and then were defined as one-fold and their expression levels in other cell lines were all compared to those in PBS-treated HME1 cells. Three independent replicates (n=3 for each replicate) were performed and results represented means of three replicates ± SD. Significant difference was determined by the One-way ANOVA, followed by Tukey's post-hoc test.
ns: no significant difference; * P < 0.05 and ** P < 0.01.  Figure S5 were used for immunoblots to measure the protein levels of ERa,ERb,Bcl2A1,Bcl2,Mcl1,and GAPDH (loading control). Three independent replicates were performed. For each lane in a replicate, three independent protein samples were mixed with equal weights (20 µg for each). One group of immunoblot images were shown. (B and C) Quantified protein levels. Protein signals in (A) were quantified using Image J software and then normalized to their corresponding loading control (GAPDH). Protein levels in PBS-treated HME1 (Ctrl) were defined as one-fold and protein levels in other cells were all compared with those in PBS-treated HME1. (B) ERa, ERb, Bcl2A1, and Bcl2. (C) Mcl1, Bcl-w, and Bcl-xL. Results represented means of three replicates ± SD. Significant difference was determined by the One-way ANOVA, followed by Tukey's post-hoc test. ns: no significant difference; * P < 0.05 and ** P < 0.01. performed and results represented means of three replicates ± SD. Significant difference was determined by the One-way ANOVA, followed by Tukey's post-hoc test. ns: no significant difference; ** P < 0.01. For immunoblots, three independent replicates were performed. For each lane in a replicate, three independent protein samples were mixed with equal weights (20 µg for each). One group of immunoblot images were shown. For quantification of protein levels, protein signals were quantified using Image J software and then normalized to their corresponding loading control (GAPDH). Protein levels in Control-KD1 cells in each background were defined as one-fold and protein levels in other cells were all compared with those in Control-KD1 (each background). Results represented means of three replicates ± SD. Significant difference was determined by the One-way ANOVA, followed by Tukey's post-hoc test. ns: no significant difference; ** P < 0.01. genes in Control-KD1 cells in each background were defined as one-fold and their expression levels in other cell lines were all compared to those in Control-KD cells (each background). Three independent replicates (n=3 for each replicate) were performed and results represented means of three replicates ± SD. Significant difference was determined by the One-way ANOVA, followed by Tukey's post-hoc test. ns: no significant difference; ** P < 0.01. For immunoblots, three independent replicates were performed. For each lane in a replicate, three independent protein samples were mixed with equal weights (20 µg for each). One group of immunoblot images were shown. For quantification of protein levels, protein signals were quantified using Image J software and then normalized to their corresponding loading control (GAPDH). Protein levels in Control-KD1 cells in each background were defined as one-fold and protein levels in other cells were all compared with those in Control-KD1 (each background). Results represented means of three replicates ± SD. Significant difference was determined by the One-way ANOVA, followed by Tukey's post-hoc test. ns: no significant difference; ** P < 0.01.  ns: no significant difference; * P < 0.05 and ** P < 0.01. performed and results represented means of three replicates ± SD. Significant difference was determined by the One-way ANOVA, followed by Tukey's post-hoc test. ns: no significant difference; ** P < 0.01. For immunoblots, three independent replicates were performed. For each lane in a replicate, three independent protein samples were mixed with equal weights (20 µg for each). One group of immunoblot images were shown. For quantification of protein levels, protein signals were quantified using Image J software and then normalized to their corresponding loading control (GAPDH). Protein levels in Control-OE1 cells were defined as one-fold and protein levels in other cells were all compared with those in Control-OE1. Results represented means of three replicates ± SD. Significant difference was determined by the One-way ANOVA, followed by Tukey's post-hoc test. ns: no significant difference; *** P < 0.001.  Figure 1D were used for detection of mRNA levels of NCOA3.

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Three independent replicates were performed and one group of representative results was shown. Significant difference was determined by the One-way ANOVA, followed by  protein signals in (B) were quantified using Image J software and then normalized to their corresponding loading control (GAPDH). Protein levels in Control-KD1 cells were defined as one-fold and protein levels in other cells were all compared with those in Control-KD1.
Results represented means of three replicates ± SD. Significant difference was determined by the One-way ANOVA, followed by Tukey's post-hoc test. ns: no significant difference; ** P < 0.01.

BCL2A1, BCL2 and MCL1 in NCOA3-p300-NF-kB-KD cells treated with or without E2
The Control-KD1, RelA-KD1, NFKB1-KD1, NCOA3-KD1, and p300-KD1 cells in T47D background were treated with 10 nM E2 or PBS for 4 h. Cells were used for ChIP assays with anti-p65, anti-p50, anti-NCOA3, anti-p300, and IgG-coupled protein G agarose. The input and output DNA samples were used for RT-qPCR analyses to determine the occupancies of NCOA3-p300-NF-kB members on the promoters of BCL2A1 (A), BCL2 (B), and MCL1 (C). Three independent replicates (n=3 for each replicate) were performed and results represented means of three replicates ± SD. Significant difference was determined by the One-way ANOVA, followed by Tukey's post-hoc test. ns: no significant difference; * P < 0.05 and ** P < 0.01.

BCL2L1 and BCL2L2 in NCOA3-p300-NF-kB-KD cells treated with or without E2
The same input and output DNA samples as in Figure S17 were used for RT-qPCR analyses to determine the occupancies of NCOA3-p300-NF-kB members on the promoters of BCL2L2 (A) and BCL2L1 (B). Three independent replicates (n=3 for each replicate) were performed and results represented means of three replicates ± SD.  The T47D cells were treated with PBS, 5 µM gossypol and 100 nM bufalin for 6 h, followed by treatment with 10 nM E2 or PBS for 4 h. Cells were used for ChIP assays with anti-NCOA3 and IgG-coupled protein G agarose. The input and output DNA samples were used for RT-qPCR analyses to determine the occupancy of NCOA3 on the promoters of BCL2A1 (A), BCL2 (B), MCL1 (C), BCL2L2 (D), and BCL1L1 (E). Three independent replicates (n=3 for each replicate) were performed and results represented means of three replicates ± SD. Significant difference was determined by the One-way ANOVA, followed by Tukey's post-hoc test. ns: no significant difference; * P < 0.05 and ** P < 0.01. For experiments in (B) and (C), three independent replicates (n=3 for each replicate) were performed and results represented means of three replicates ± SD. Significant difference was determined by the One-way ANOVA, followed by Tukey's post-hoc test. * P < 0.05.
(D) Tumor volumes. T47D cells were injected into female nude mice (n=60 for each cell line), followed by implantation with or without 0.18 mg E2 pellet. Mice were randomly grouped into three groups (Control, gossypol, and bufalin) when tumor volumes reached ~150 mm 3 . The Control group mice were injected with PBS. The gossypol groups of mice were injected with 50 mg/kg gossypol and the bufalin groups of mice were injected with 1.5 mg/kg bufalin. Tumor volumes were measured at 5-day intervals for 30 days. Three independent replicates (n=10 for each replicate) were performed and results represented means of three replicates ± SD. Significant difference was determined by the One-way ANOVA, followed by Tukey's post-hoc test. At each time point, comparisons of T47D vs.
T47D+0.18 mg E2 (red asterisks); T47D vs. 1.5 mg/kg bufalin; T47D vs. 50 mg/kg 100 nM bufalin+10 nM E2 at a density of 1 ´ 10 3 cells/well in 2 mL DMEM containing 10% charcoal stripped FBS. Cell colonies were fixed with 5% glutaraldehyde at 37°C for 10 min, followed by staining with 0.1% crystal violet. Three independent replicates (n=3 for each replicate) were performed and one group of images were shown. (B) Representative images of cell invasion. The same cells as in (A) were seeded in the upper compartment of the Nunc Polycarbonate Cell Culture Inserts at a density of 1 ´ 10 5 cells/well in the conditions of 10 nM E2 and PBS (control). After incubation at 37°C for 48 h, invaded cells in the lower insert were fixed with 5% glutaraldehyde and stained with 0.1% crystal violet.
Three independent replicates (n=3 for each replicate) were performed and one group of images were shown. Bars=50 µm.